Identification and Characterization of an Operon, \u3ci\u3emsaABCR\u3c/i\u3e, That Controls Virulence and Biofilm Development in \u3ci\u3eStaphlococcus aureus\u3c/i\u3e

Background Community-acquired, methicillin-resistant Staphylococcus aureus strains often cause localized infections in immunocompromised hosts, but some strains show enhanced virulence leading to severe infections even among healthy individuals with no predisposing risk factors. The genetic basis for this enhanced virulence has yet to be determined. S. aureus possesses a wide variety of virulence factors, the expression of which is carefully coordinated by a variety of regulators. Several virulence regulators have been well characterized, but others have yet to be thoroughly investigated. Previously, we identified the msa gene as a regulator of several virulence genes, biofilm development, and antibiotic resistance. We also found evidence of the involvement of upstream genes in msa function. Results To investigate the mechanism of regulation of the msa gene (renamed msaC), we examined the upstream genes whose expression was affected by its deletion. We showed that msaC is part of a newly defined four-gene operon (msaABCR), in which msaC is a non-protein-coding RNA that is essential for the function of the operon. Furthermore, we found that an antisense RNA (msaR) is complementary to the 5′ end of the msaB gene and is expressed in a growth phase-dependent manner suggesting that it is involved in regulation of the operon. Conclusion These findings allow us to define a new operon that regulates fundamental phenotypes in S. aureus such as biofilm development and virulence. Characterization of the msaABCR operon will allow us to investigate the mechanism of function of this operon and the role of the individual genes in regulation and interaction with its targets. This study identifies a new element in the complex regulatory circuits in S. aureus, and our findings may be therapeutically relevant

MsaB and CodY Interact to Regulate \u3ci\u3eStaphylococcus aureus\u3c/i\u3e Capsule in a Nutrient-Dependent Manner

Staphylococcus aureus has a complex regulatory network for controlling the production of capsule polysaccharide. In S. aureus, capsule production is controlled by several regulators in response to various environmental stimuli. Previously, we described MsaB as a new regulator that specifically binds to the cap promoter in a growth-phase or nutrient-dependent manner. In addition to MsaB, several other regulators have also been shown to bind the same region. In this study, we examined the interactions between MsaB and other nutrient-sensing regulators (CodY and CcpE) with respect to binding to the cap promoter in a nutrient-dependent manner. We observed that msaABCR and ccpE interact in a complex fashion to regulate capsule production. However, we confirmed that ccpE does not bind cap directly. We also defined the regulatory relationship between msaABCR and CodY. When nutrients (branched chain amino acids) are abundant, CodY binds to the promoter region of the cap operon and represses its transcription. However, when nutrient concentrations decrease, MsaB, rather than CodY, binds to the cap promoter. Binding of MsaB to the cap promoter activates transcription of the cap operon. We hypothesize that this same mechanism may be used by S. aureus to regulate other virulence factors

Delineating the Role of the msaABCR Operon in Staphylococcal Overflow Metabolism

Staphylococcus aureus is an important human pathogen that can infect almost every organ system, resulting in a high incidence of morbidity and mortality. The msaABCR operon is an important regulator of several staphylococcal phenotypes, including biofilm development, cell wall crosslinking, antibiotic resistance, oxidative stress, and acute and chronic implant-associated osteomyelitis. Our previous study showed that, by modulating murein hydrolase activity, the msaABCR operon negatively regulates the proteases that govern cell death. Here, we report further elucidation of the mechanism of cell death, which is regulated by the msaABCR operon at the molecular level in the USA300 LAC strain. We showed that deletion of msaABCR enhances weak-acid-dependent cell death, because, in the biofilm microenvironment, this mutant strain consumes glucose and produces acetate and acetoin at higher rates than wild-type USA300 LAC strain. We proposed the increased intracellular acidification leads to increased cell death. MsaB, a dual-function transcription factor and RNA chaperone, is a negative regulator of the cidR regulon, which has been shown to play an important role in overflow metabolism and programmed cell death during biofilm development in S. aureus. We found that MsaB binds directly to the cidR promoter, which represses expression of the cidR regulon and prevents transcription of the cidABC and alsSD operons. In addition, we observed that pyruvate induced expression of the msaABCR operon (MsaB). The results reported here have enabled us to decipher the role of the msaABCR operon in staphylococcal metabolic adaption during biofilm development

The \u3ci\u3emsaABCR\u3c/i\u3e Operon Regulates Persister Formation by Modulating Energy Metabolism in \u3ci\u3eStaphylococcus aureus\u3c/i\u3e

Staphylococcus aureus is a major human pathogen that causes chronic, systemic infections, and the recalcitrance of these infections is mainly due to the presence of persister cells, which are a bacterial subpopulation that exhibits extreme, yet transient, antibiotic tolerance accompanied by a transient halt in growth. However, upon cessation of antibiotic treatment, a resumption in growth of persister cells causes recurrence of infections and treatment failure. Previously, we reported the involvement of msaABCR in several important staphylococcal phenotypes, including the formation of persister cells. Additionally, observations of the regulation of several metabolic genes by the msaABCR operon in transcriptomics and proteomics analyses have suggested its role in the metabolic activities of S. aureus. Given the importance of metabolism in persister formation as our starting point, in this study we demonstrated how the msaABCR operon regulates energy metabolism and subsequent antibiotic tolerance. We showed that deletion of the msaABCR operon results in increased tricarboxylic acid (TCA) cycle activity, accompanied by increased cellular ATP content and higher NADH content in S. aureus cells. We also showed that msaABCR (through MsaB) represses the ccpE and ndh2 genes, thereby regulating TCA cycle activity and the generation of membrane potential, respectively. Together, the observations from this study led to the conclusion that msaABCR operon deletion induces a metabolically hyperactive state, leading to decreased persister formation in S. aureus

Evaluation of Antioxidant Capacities and Total Polyphenols in Various Edible Parts of Capparis spinosa L. Collected from Trans-Himalayas

The phytochemical screening, antioxidant capacity, and total polyphenols in the methanolic extract of leaves, flower buds, roots and fruits of Capparis spinosa collected from trans-Himalayan region of Ladakh were assessed in an effort to corroborate its medicinal and culinary potential. Highest DPPH and ABTS radical scavenging activity were observed in the leaves and least in dried fruit samples, even FRAP assay also illustrated the same trend. IC50 values of DPPH assay was highly correlated with that of ABTS (R2=0.9084) and FRAP assay (R2=0.9771). However, IC50 value of ABTS was reasonably correlated with FRAP assay (R2=0.5838). The highest phenolic and flavonoid content was recorded in the leaf samples (24.78 and 5.69 mg GAE/g DW respectively), whereas it was lowest in the dried fruit samples (4.07 mg quercetin equivalent/g DW and nil, respectively). The total phenolic contents were highly correlated with IC50 value of ABTS (R2=0.9084), DPPH (R2=0.9388) and FRAP value (R2=0.9618). But, total flavonoid contents were highly correlated with ABTS (R2=0.7449), DPPH (R2=0.8791) and FRAP values (R2=0.9588). Thus, this study has validated the medicinal potential of all the edible parts of the C. spinosa